Heteroaromatic NMDA receptor modulators and uses thereof

ABSTRACT

Provided is 5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one and pharmaceutically acceptable salts thereof, and their uses in the treatment of psychiatric, neurological, and neurodevelopmental disorders, as well as diseases of the nervous system.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/919,136 filed Jul. 2, 2020, which is a continuation of U.S.application Ser. No. 16/678,806 filed Nov. 8, 2019, which is acontinuation of U.S. application Ser. No. 16/530,274 filed Aug. 2, 2019,which claims the benefit of priority to U.S. Provisional Application No.62/714,100 filed Aug. 3, 2018, the entire contents of each of which areincorporated herein by reference.

BACKGROUND

Diseases of the nervous system are collectively the leading cause ofhuman disability, as measured by the global burden of disease. Eventhose major diseases of the nervous system for which treatments havebeen approved by health authorities, including psychiatric diseases suchas Schizophrenia, neurological diseases such as Alzheimer's Disease, andneurodevelopmental disorders, such as Attention Deficit andHyperactivity Disorder, are poorly managed because approved treatmentshave limited efficacy and serious side effects, leaving a significantburden of unmet medical need. In addition, there are many major and rarenervous system disorders for which no treatments are approved, such asthe neurodevelopmental disorders of the Autism Spectrum, and manyintellectual disability disorders, and which are therefore associatedwith profound unmet medical need.

The N-methyl-D-aspartate-(NMDA) subtype of ligand-gated ion channelreceptors are a diverse family of glutamate receptors widely accepted tomediate synaptic transmission, key mechanisms of synaptic plasticity,and dynamic neuronal network connectivity required for normal nervoussystem development and function.

The NMDA receptor is composed of four protein subunits, two GluN1subunits and two GluN2 subunits. The GluN1 subunit is derived from asingle gene (GRIN1), is ubiquitously expressed throughout the nervoussystem, and is common to all NMDA receptors. Four different GluN2subunits, GluN2A-D, are derived from separate genes (GRIN2A-D) that aredifferentially expressed in different regions of the nervous system andby distinct populations of neurons within a particular region. A GluN3subunit has also been identified, but its function is less wellunderstood. Furthermore, individual neurons may express more than oneGluN2 subunit and individual NMDA receptors expressed by such neuronsmay contain two of the same GluN2 subunits (for example, 2GluN2Bsubunits) or two different subunits (one GluN2A and one GluN2B subunit).In addition, all NMDA receptor subunits are expressed as diverse mRNAsplice variants. Thus, native nervous system NMDA receptors are highlydiverse in their composition.

The study of the molecular basis of NMDA receptor function continues tobe an area of importance. As glutamate is the major excitatoryneurotransmitter, dysfunction of glutamate neurotransmission and NMDAreceptor-dependent mechanisms of synaptic transmission, plasticity, andneuronal network connectivity are broadly implicated in diseases of thenervous system. Accordingly, compounds that are capable of modulatingNMDA receptors may be useful for the treatment of nervous systemdisorders and diseases, for example, schizophrenia, Alzheimer's disease,attention deficit and hyperactivity disorder, and autism.

SUMMARY

Provided herein is Compound 1,5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one,having the following structure:

Compound 1 is a heterocyclic pyrrolopyrimidinone analogue whichmodulates NMDA receptors (e.g., positive allosteric modulation of NMDAreceptors) and may be used for treating psychiatric, neurological, andneurodevelopmental disorders, as well as diseases of the nervous system.

Heterocyclic NMDA receptor modulators have been previously described ine.g., WO 2017/100591. In WO 2017/100591, a wide range of heterocycliccores including pyrrolopyrazinones, thienopyridinones,imidazopyrazinones, pyrrolopyridinones, and pyrrolopyrimidinones aredisclosed. The potency of the compounds in WO 2017/100591 was evaluatedon the basis of the compounds' ability to reverse the suppression of theCa²⁺ response mediated by Ro 25-6981, a potent and selective antagonistof NMDA glutamate receptors containing the NR2B subunit, and5,7-dichlorokynurenic acid (DCKA), a selective NMDA receptor antagonistacting at the glycine site of the NMDA receptor complex. Potency in theRo 25-6981/DCKA assay was quantified by % response recovered (as showne.g., in Table 49 of WO 2017/100591) and/or by % maximum measuredpotentiation. Of the most potent twenty-one analogs exemplified in WO2017/100591 in the Ro 25-6981/DCKA assay and ranked by % responserecovered, none were shown to be pyrrolopyrimidinone analogues.

There are forty-eight pyrrolo[2,3-d]pyrimidin-4-ones exemplified in WO2017/100591. This pyrrolo[2,3-d]pyrimidin-4-one core is identical to theheterocyclic core of Compound 1. The most potent exemplifiedpyrrolo[2,3-d]pyrimidin-4-one based on % response recovered in the Ro25-6981/DCKA assay exemplified in WO 2017/100591 was Example 174 (91%response recovered). See e.g., Table 49 of WO 2017/100591. Yet, as shownin Table 1 below, Compound 1 is approximately three-fold more potentthan Example 174 in the disclosed oocyte NR2B PAM potentiation assay.Another exemplified pyrrolo[2,3-d]pyrimidin-4-one in WO 2017/100591 isExample 181. This compound was determined to be the most potentexemplified pyrrolo[2,3-d]pyrimidin-4-one based on the maximum measuredpotentiation (%) in the Ro 25-6981/DCKA assay. See Table 1 below for acomparison of Examples 174 and 181 in these two potency readouts fromthis Ro 25-6981/DCKA assay. Similar to the results with Example 174,Compound 1 was again found to be more potent (approximately 2.5-fold) inthe disclosed oocyte NR2B PAM potentiation assay. Taking together, theseresults evidence the clinical potency advantage of Compound 1 over otherpyrrolo[2,3-d]pyrimidin-4-one based scaffolds.

In terms of structurally related analogues, WO 2017/100591 discloses aconstitutional isomer of Compound 1 (i.e., Example 436) as well as a onenitrogen variant (i.e., Example 285). Despite these structuralsimilarities, Compound 1 was found to possess superior aqueoussolubility, microsomal stability, and/or pharmacokinetic properties ascompared to these two compounds. Table 2, for example, shows thatCompound 1 has approximately a 10-fold increase in aqueous solubilityover Example 436, the constitutional isomer of Compound 1. Compound 1was found to have excellent cell permeability (32×10⁻⁶ cm/s for A→B andB→A) and was not a substrate for efflux in MDR1-transfected MDCK cells(efflux ratio=0.99). See Table 3. Table 4 shows that Compound 1demonstrates significantly improved rat liver microsomal stabilityversus Example 285 (t_(1/2)>120 min versus 37.8 min, respectively) andalso demonstrates significantly improved in vivo clearance in rat versusExample 285 (IV clearance=7.4 mL/min/kg versus 22.2 mL/min/kg) andseveral other analogs exemplified in WO 2017/100591.

The data above establishes numerous clinical advantages of Compound 1,thereby providing a solution to finding alternative NMDA modulatorshaving e.g., improved potency, enhanced solubility, favorable microsomalstability and in vivo clearance, and excellent cell permeability.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the cognitive enhancing effect of oral Compound 1(0.3, 1.0, and 3.0 mg/kg) against phencyclidine (PCP)-induced cognitiveimpairment in the rat Novel Object Recognition (NOR) test.

FIG. 2 illustrates the results from Compound 1 in a mismatch negativityefficacy model.

DETAILED DESCRIPTION

1. Compounds

Provided herein is a compound having the following structure:

or a pharmaceutically acceptable salt thereof2. Definitions

As used herein the terms “subject” and “patient” may be usedinterchangeably, and means a mammal in need of treatment, e.g.,companion animals (e.g., dogs, cats, and the like), farm animals (e.g.,cows, pigs, horses, sheep, goats and the like) and laboratory animals(e.g., rats, mice, guinea pigs and the like). Typically, the subject isa human in need of treatment.

Pharmaceutically acceptable salts as well as the neutral forms of thecompounds described herein are included. For use in medicines, the saltsof the compounds refer to non-toxic “pharmaceutically acceptable salts.”Pharmaceutically acceptable salt forms include pharmaceuticallyacceptable acidic/anionic or basic/cationic salts. Pharmaceuticallyacceptable basic/cationic salts include, the sodium, potassium, calcium,magnesium, diethanolamine, n-methyl-D-glucamine, L-lysine, L-arginine,ammonium, ethanolamine, piperazine and triethanolamine salts.Pharmaceutically acceptable acidic/anionic salts include, e.g., theacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, carbonate,citrate, dihydrochloride, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrobromide, hydrochloride, malate, maleate,malonate, mesylate, nitrate, salicylate, stearate, succinate, sulfate,tartrate, and tosylate.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier, adjuvant, or vehicle that does not destroy the pharmacologicalactivity of the compound with which it is formulated. Pharmaceuticallyacceptable carriers, adjuvants or vehicles that may be used in thecompositions described herein include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium tri silicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

“Pharmaceutically acceptable” means molecular entities and compositionsthat do not produce an adverse, allergic or other untoward reaction whenadministered to an animal, or a human, as appropriate.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, reducing the likelihood of developing, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed, i.e., therapeutic treatment.In other embodiments, treatment may be administered in the absence ofsymptoms. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors),i.e., prophylactic treatment. Treatment may also be continued aftersymptoms have resolved, for example to prevent or delay theirrecurrence.

The term “effective amount” or “therapeutically effective amount”includes an amount of a compound described herein that will elicit abiological or medical response of a subject e.g., a dosage of between0.01-100 mg/kg body weight/day.

“Compound 1” and“5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one”are used interchangeably and each refer to the compound having thefollowing formula:

“PAM” refers to “positive allosteric modulator”.

3. Uses, Formulation and Administration

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereofand compositions described herein are useful in treating diseases and/ordisorders associated with the activity of NMDA receptors. Such diseasesand/or disorders include e.g., psychiatric, neurological, andneurodevelopmental disorders, as well as diseases of the nervous system.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for modulating theactivity of the NMDA receptor.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for treatingschizophrenia, Alzheimer's disease, attention deficit and hyperactivity,autism, and other nervous system-associated conditions.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for treatingschizophrenia, including positive, negative, and cognitive symptoms.Schizophrenia is a debilitating mental disorder encompassing threesymptom domains: positive (psychosis, hallucination, delusions),negative (withdrawal), and cognitive (global reduction in cognitiveability). Positive symptoms of schizophrenia typically emerge early inadulthood and are treated with antipsychotic medications. However,cognitive deficits are severe, emerge in the adolescent prodromal stage,are resistant to antipsychotic therapy, and are the leading cause oflifetime disability as measured by impaired global function (inabilityto live independently, unemployment, etc). NMDA receptor hypofunction isthe leading hypothesis for the cause of schizophrenia. This hypothesisis supported by substantial clinical evidence including clinicalpharmacology, electrophysiology, imaging, cognition, computationalneuroscience, neuroanatomical studies, and genetics. In particular,several lines of evidence implicate hypofunction of NMDA receptors inschizophrenia. See Frank S. Menniti, Craig W. Lindsley, P. Jeffrey Conn,Jayvardhan Pandit, Panayiotis Zagouras, and Robert A. Volkmann,Allosteric Modulators for the Treatment of Schizophrenia: TargetingGlutamatergic Networks. Curr Top Med Chem. 2013; 13(1): 26-54.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for improvingcognitive and global function, and/or preventing the onset ofschizophrenia e.g., in people at risk of developing schizophrenia.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for treatingcognitive and emotional deficits and other symptoms associated withexemplary psychiatric disorders including major depressive disorder, andincluding but not limited to those suffering from schizoaffectivedisorder, bipolar disorder, obsessive-compulsive disorder, dysphobicdisorder, dysthymic disorder, psychotic depression, post-traumaticstress disorder, and other anxiety disorders. For example, providedherein are methods of treating attention deficit disorder, ADHD(attention deficit hyperactivity disorder), schizophrenia, anxiety,amelioration of opiate, nicotine and/or ethanol addiction (e.g., methodof treating such addiction or ameliorating the side effects ofwithdrawing from such addiction), spinal cord injury, diabeticretinopathy, traumatic brain injury, and/or post-traumatic stresssyndrome in a patient in need thereof, that includes administeringCompound 1, or a pharmaceutically acceptable salt thereof, or acomposition thereof.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for treatingcognitive and emotional deficits and other symptoms resulting fromneurological diseases, including but not limited to a patient sufferingfrom mild cognitive impairment or any form of dementia, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, multiple sclerosis, and seizure disorders.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for treatingdysfunction caused by neurodevelopmental disorders, e.g., abnormal braindevelopment, including but not limited to Rett Syndrome, AttentionDeficit and Hyperactivity Disorder, autism and autism spectrum disorderssuch as Phelan-McDermid Syndrome, and other forms of intellectualdisability such as Fragile X syndrome, tuberous sclerosis,Smith-Lemli-Opitz Syndrome, Down syndrome and childhood epilepsies orepilepsy/aphasia spectrum disorders such as Benign partial Epilepsy ofchildhood with CentroTemporal Spikes (BECTS) or Landau-Kleffner Syndrome(LKS). A method is also provided to treat patients suffering fromabnormal brain function resulting from infections of the central nervoussystem, exposure to toxic agents or other xenobiotics or naturallyoccurring toxins, and/or autoimmune disorders including, but not limitedto anti-NMDA receptor encephalitis.

In another aspect, Compound 1 or a pharmaceutically acceptable saltthereof and compositions described herein are useful for treatingsubjects having NMDA receptor hypofunction.

Provided herein is a method of treating a subject having a disease,disorder, or condition described herein comprising administering to thesubject a therapeutically effective amount of Compound 1, or apharmaceutically acceptable salt thereof, or a composition thereof.

Also provided is the use of Compound 1, or a pharmaceutically acceptablesalt thereof, or a composition thereof, for the manufacture of amedicament for treating a disease, disorder, or condition describedherein.

Also provided is Compound 1, or a pharmaceutically acceptable saltthereof, or a composition thereof, for use in treating a disease,disorder, or condition described herein.

In one aspect, provided are pharmaceutically acceptable compositionscomprising Compound 1; and a pharmaceutically acceptable carrier. Thesecompositions can be used to treat one or more of the diseases,disorders, and conditions described above.

The disclosed compositions may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. Liquid dosage forms,injectable preparations, solid dispersion forms, and dosage forms fortopical or transdermal administration of a compound are included herein.

The amount of Compound 1 that may be combined with carrier materials toproduce a composition in a single dosage form will vary depending uponthe patient to be treated and the particular mode of administration. Insome embodiments, provided compositions may be formulated so that adosage of between 0.01-100 mg/kg body weight/day of the providedcompound, such as e.g., 0.1-100 mg/kg body weight/day, can beadministered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the judgment of thetreating physician, and the severity of the particular disease beingtreated. The amount of a provided compound in the composition will alsodepend upon the particular compound in the composition.

Combination therapies using a therapeutically effective amount ofCompound 1, or a pharmaceutically acceptable salt thereof, and aneffective amount of one or more additional pharmaceutically activeagents is also included herein. In one aspect, for example, provided isthe use of Compound 1, or a pharmaceutically acceptable salt thereof,and an effective amount of one or more atypical antipsychotics to treata disorder or disease described herein. Atypical antipsychotics include,e.g., lurasidone, quetiapine, olanzapine, asenapine, risperidone,ziprasidone, clozapine, mel perone, cariprazine, aripiprazole,pimavanserin, ITI-007, RP506, and remoxipride.

EXEMPLIFICATION

The representative examples that follow are intended to help illustratethe present disclosure, and are not intended to, nor should they beconstrued to, limit the scope of the invention.

Example 1 Preparation of5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-411-pyrrolo[2,3-d]pyrimidin-4-oneCompound 1

Step-1: Preparation of 4,6-dichloro-5-(2,2-diethoxyethyl)pyrimidine (2)

To a solution of 2-(4,6-dichloropyrimidin-5-yl)acetaldehyde (1, 5 g,26.17 mmol) in ethanol (250 mL), ammonium chloride (2.11 g, 39.52 mmol)was added and the reaction mixture was refluxed for 20 h. The reactionmixture was concentrated; the residue was diluted with water (50 mL) andextracted with ethyl acetate (2×100 mL). The combined organic layer wasdried over anhydrous sodium sulphate, filtered and concentrated toafford the title compound 4,6-dichloro-5-(2,2-diethoxyethyl)pyrimidine(2, 6.9 g, crude) as colorless oil. Calculated (M+H): 265.04; Found(M+H): 265

Step-2: Preparation of4-chloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine (3)

To a solution of 4,6-dichloro-5-(2,2-diethoxyethyl)pyrimidine (2, 6.9 g,26.02 mmol) and triethylamine (3.63 mL, 26.02 mmol) in ethanol (150 mL),cyclopropylamine (2 mL, 28.62 mmol) was added and the reaction mixturewas refluxed for 10 h. The reaction mixture was evaporated under vacuum;the residue was diluted with water (100 mL) and extracted with ethylacetate (2×200 mL). The combined organic layer was dried over anhydroussodium sulphate, filtered and concentrated. The residue was dissolved intetrahydrofuran (150 mL), 2M hydrochloric acid (75 mL) was added and thereaction mixture was refluxed for 1 h. The reaction mixture wasconcentrated, the residue was dissolved in water (100 mL), basified topH 10 with sodium hydroxide solution and extracted with ethyl acetate(2×200 mL). The combined organic layer was dried over anhydrous sodiumsulphate, filtered and concentrated to afford the title compound4-chloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine (3, 5 g, crude) asbrownish semi-solid. Calculated (M+H): 194.04; Found (M+H): 194

Step-3: Preparation of7-cyclopropyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (4)

A solution of 4-chloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine (3, 19g, 98.44 mmol) in dioxane (400 mL) and 2N sodium hydroxide solution (400mL) was heated at 100° C. for 16 h. The reaction mixture wasconcentrated to remove dioxane. The aqueous residue was diluted withwater (−200 mL) and acidified to pH ˜4-6 using 1.5N hydrochloric acidsolution. The precipitated solid was filtered, washed with hexane anddried under suction to afford the title compound7-cyclopropyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (4, 14.93 g,87% yield) as a brownish solid. Calculated (M+H): 176.07; Found (M+H):176

Step-4: Preparation of ethyl2-(7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)acetate(5)

To a solution of7-cyclopropyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (4, 2.7 g,15.41 mmol) in acetone (60 mL) were added ethyl 2-bromoacetate (5.1 g,30.8 mmol) and potassium carbonate (6.2 g, 46.2 mmol) at roomtemperature and the reaction mixture was stirred at 55° C. for 4 h. Thereaction mixture was cooled to room temperature and filtered. The solidwas washed with ethyl acetate (100 mL) and the combined filtrate wasevaporated to get crude product, which was purified by silica gel columnchromatography using 40% ethyl acetate in hexane to afford the titlecompound ethyl2-(7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)acetate(5, 3.5 g, 87% yield) as off-white solid. Calculated (M+H): 262.11;Found (M+H): 262

Step-5: Preparation of ethyl2-(5-bromo-7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)acetate(6)

To a stirred solution of ethyl2-(7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)acetate(5, 3 g, 11.4 mmol) in N,N-dimethyl formamide (200 mL),N,O-(bis-trimethylsilyl)acetamide (5.1 g, 25 mmol) andN-bromosuccinimide (2.02 g, 11.4 mmol) were added and the reactionmixture was stirred at room temperature for 2 h. The reaction mixturewas quenched with cold water (400 mL) and extracted with ethyl acetate(2×200 mL). The combined organic layer was dried over anhydrous sodiumsulphate, filtered and concentrated. The crude product was purified bysilica gel column chromatography using 35% ethyl acetate in hexane toafford the title compound ethyl2-(5-bromo-7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)acetate(6, 2.1 g, 54% yield) as an off white solid. Calculated (M+H): 340.02;Found (M+H): 340

Step-6: Preparation of2-(5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)aceticacid (7)

To a solution of ethyl2-(5-bromo-7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)acetate(6, 7.5 g, 22.04 mmol) and (3-chloro-4-fluorophenyl)boronic acid (5.76g, 33.07 mmol) in 1,4-dioxane:water mixture (250 mL, 4:1), potassiumcarbonate (9.15 g, 66.14 mmol) was added. The reaction mixture waspurged with argon for 20 min. Then[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (0.9 g, 1.10 mmol) was added and the reactionmixture was stirred at 100° C. for 16 h. The reaction mixture wasfiltered through celite bed, the filtrate was diluted with water (100mL) and washed with ethyl acetate (2×200 mL). The organic layer wasdiscarded. The aqueous layer was acidified with 1.5N hydrochloric acid,the precipitated solid was filtered and dried under suction to affordthe title compound2-(5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)aceticacid (7, 6 g, crude) as an off-white solid. Calculated (M+H): 362.06;Found (M+H): 362.1

Step-7: Preparation of5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one

To a stirred solution of2-(5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)aceticacid (7, 1.8 g, 4.9 mmol) in dichloromethane (70 mL) were addedtriethylamine (1.36 mL, 9.9 mmol) and 3-fluoro-3-methylazetidinehydrochloride (1.24 g, 9.9 mmol) at room temperature. The reactionmixture was stirred for 10 min, then propylphosphonic anhydride solution(T₃P) (6.33 mL, 9.9 mmol, 50% in ethyl acetate) was added at 0° C. andreaction mixture was stirred at room temperature for 16 h. The reactionmixture was diluted with water (50 mL) and extracted withdichloromethane (3×70 mL). The combined organic layer was dried overanhydrous sodium sulfate, filtered and evaporated to get crude product,which was purified by silica gel column chromatography using 4% methanolin dichloromethane to afford the title compound5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(Compound 1, 1.1 g, 51% yield) as a white solid. Calculated (M+H):433.12; Found (M+H): 433.1, ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 8.26 (d,J=6 Hz, 1H), 8.15 (s, 1H), 7.93-7.91 (m, 1H), 7.58 (s, 1H), 7.35 (t,J=8.8 Hz, 1H), 4.67 (s, 2H), 4.45-4.32 (m, 2H), 4.05-3.97 (m, 2H),3.64-3.59 (m, 1H). 1.61 (d, J=22 Hz, 3H), 1.10-1.00 (m, 4H). HPLCpurity: 99.28%.

Example 2 Alternative Preparation of5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (Compound 1)

Step 1: Preparation of5-bromo-4-chloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine 10:

To a solution of 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine 9 (10.0 g,43.10 mmol) in 1,2-dichloroethane (100.0 mL) were addedcyclopropylboronic acid (7.41 g, 86.20 mmol), sodium carbonate (10.0 g,86.20 mmol) and 2,2′-bipyridyl (6.70 g, 43.53 mmol). Then oxygen waspurged into the reaction mixture for 30 min and copper acetate (8.21 g,45.25 mmol) was added. The suspension was stirred at 80° C. for 18 h andafter completion the reaction mixture was quenched with 1N hydrochloricacid at room temperature (up to pH=1.0). The solution was extracted withdichloromethane (4×100 mL) and the combined organic layer was dried overanhydrous sodium sulphate. The solution was concentrated and the residuewas purified by silica gel column chromatography (using 10% ethylacetate in hexane) to afford the title compound5-bromo-4-chloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine 10 asoff-white solid. Yield: 8.00 g, 68.4%. MS (ESI): m/z 271.94 [M+1]⁺.

Step 2: Synthesis of5-bromo-7-cyclopropyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (11)

A solution of 5-bromo-4-chloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine10 (8.00 g, 29.52 mmol) in dioxane (40.00 mL) and 2N sodium hydroxidesolution (40.00 mL) was heated at 95° C. for 18 hours. After completion,the reaction mixture was concentrated to remove dioxane. The aqueousresidue was diluted with water (about 50 mL) and acidified to pH ofabout 3 using 1N hydrochloric acid solution. The precipitated solid wasfiltered, washed with hexane and dried under suction to afford the titlecompound5-bromo-7-cyclopropyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 11 asoff white solid. Yield: 5.80 g, 77.70%. MS (ESI) m/z 254.05 [M+1]⁺.

Synthesis of Intermediate2-bromo-1-(3-fluoro-3-methylazetidin-1-yl)ethan-1-one (12)

To a stirred solution of compound A (44.0 g, 352 mmol) in DCM (400 mL)was added NaOH (1.0 M solution, 14.0 g, 352 mmol) and stirred thebiphasic solution at room temperature for 30 min. The solution wasseparated and the aqueous phase was extracted with DCM (50 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄ and taken into aRBF. The solution was cooled to −10° C. and then bromoacetyl bromide B(106.5 mL, 528 mmol) was added slowly (to maintain the internaltemperature at −5° C.) and stirred at same temperature for 2 h. Aftercompletion, the reaction mixture was quenched with cold sat NaHCO₃solution and both layers were separated. The organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. The oilyresidue was quickly flashed through silica gel column (20-30% of EtOAcin hexanes) to afford2-bromo-1-(3-fluoro-3-methylazetidin-1-yl)ethan-1-one 12 as light brownoil. Yield: 53.0 g (73%). MS (ESI) m/z 209.98 [M+1]⁺.

Step 3: Synthesis of5-bromo-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one13

To a solution of5-bromo-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one11 (11.0 g, 43 mmol) in acetone (110 mL) were added2-bromo-1-(3-fluoro-3-methylazetidin-1-yl)ethan-1-one 12 (10.9 g, 52mmol) and K₂CO₃ (11.8 g, 86 mmol) at room temperature. The reactionmixture was stirred at 60° C. for 16 h. After completion, the reactionmixture was cooled to room temperature, filtered through Celite® andwashed with acetone (500 mL). The combined filtrate was evaporated toget the crude product, which was purified by washing thoroughly withdiethyl ether to afford title compound5-bromo-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one13 as a light brown solid. Yield: 14.9 g (89.7%). MS (ESI) m/z 294.23[M+1]⁺.

Step 4: Synthesis of5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(Compound 1)

To a solution of5-bromo-7-cyclopropyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 13(0.5 g, 1.3 mmol) in 1,4-dioxane (4.0 mL) were added(3-chloro-4-fluorophenyl)boronic acid (0.22 g, 1.3 mmol) and K₂CO₃ (0.36g, 2.6 mmol, 2 M solution). The reaction mixture was first degassed withargon for 10 min and then PdCl₂(dppf)DCM (0.047 g, 0.065 mmol) was addedunder argon. The reaction mixture was stirred at 80° C. for 6 h. Aftercompletion, the reaction mixture was cooled to room temperature,filtered and washed with DCM (30 mL). The combined filtrate wasevaporated to get the crude product, which was purified by silica gelcolumn chromatography (5-6% methanol in DCM) and washed with THF toafford the title compound5-(3-chloro-4-fluorophenyl)-7-cyclopropyl-3-(2-(3-fluoro-3-methylazetidin-1-yl)-2-oxoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(Compound 1) as a light gray solid. Yield: 0.170 g (30%). MS (ESI) m/z433.23 [M+1]⁺.

Biological Assays

As shown by the data presented below, Compound 1 is a potent positiveallosteric modulator across all of the NR2A, NR2B, NR2C and NR2D NMDAsubtypes.

Oocyte 2EVC NR2A PAM Activity

In 2-electrode voltage clamp (2EVC) testing of oocytes expressing hNR2Aand activated with 7 μM glutamate and 13 μM glycine, the potentiation ofcurrent (100% potentiation is equivalent to doubling of current inducedby glutamate/glycine) and potency of Compound 1 was measured. EC₅₀refers to the concentration of a compound that gives half-maximalresponse.

Oocyte 2EVC Oocyte 2EVC NR2A PAM: Oocyte 2EVC NR2A PAM: ConcentrationNR2A PAM: Avg Max % Required for Current Avg EC₅₀ (nM) Potentiation (%)Doubling (nM) 439 350 220

Oocyte 2EVC NR2C PAM Activity

Description: In 2-electrode voltage clamp (2EVC) testing of oocytesexpressing hNR2C and activated with 10 μM glutamate and 10 μM glycine,the potentiation of current (100% potentiation is equivalent to doublingof current induced by glutamate/glycine) and potency of Compound 1 wasmeasured. EC₅₀ refers to the concentration of a compound that giveshalf-maximal response.

Oocyte 2EVC Oocyte 2EVC NR2C PAM: Oocyte 2EVC NR2C PAM: ConcentrationNR2C PAM: Avg Max % Required for Current Average EC₅₀ (nM) Potentiation(%) Doubling (nM) 1203 1800 120

Oocyte 2EVC NR2D PAM Activity

Description: In 2-electrode voltage clamp (2EVC) testing of oocytesexpressing hNR2D and activated with 10 μM glutamate and 10 μM glycine,the potentiation of current (100% potentiation is equivalent to doublingof current induced by glutamate/glycine) and potency of Compound 1 wasmeasured. EC₅₀ refers to the concentration of a compound that giveshalf-maximal response.

Oocyte 2EVC Oocyte 2EVC NR2D PAM: Oocyte 2EVC NR2D PAM: ConcentrationNR2D PAM: Avg Max % Required for Current Average EC₅₀ (nM) Potentiation(%) Doubling (nM) 1887 1700 165

Oocyte 2EVC NR2B PAM Activity

In 2-electrode voltage clamp(2EVC) testing of oocytes expressing hNR2Band activated with 5 μM glutamate and 3 μM glycine, the potentiation ofcurrent (100% potentiation is equivalent to doubling of current inducedby glutamate/glycine) and potency of Compound 1 was measured. EC₅₀refers to the concentration of a compound that gives half-maximalresponse.

Oocyte 2EVC Oocyte 2EVC NR2B PAM: Oocyte 2EVC NR2B PAM: ConcentrationNR2B PAM: Avg Max % Required for Current Avg EC₅₀ (nM) Potentiation (%)Doubling (nM) 446 478 160

As shown in Table 1 below, Compound 1 was compared against Example 174(the most potent exemplified compound tested in the Ro 25-6981/DCKAassay quantified by % response recovered) and Example 181 (the mostpotent exemplified compound tested in the Ro 25-6981/DCKA assayquantified by % maximum measured potentiation). As shown in the table,Compound 1 is approximately three-fold and 2.5-fold more potent in theoocyte NR2B PAM potentiation assay than Example 174 and Example 181,respectively. This improvement is advantageous because higher potency inthe oocyte NR2B PAM potentiation assay is, in one aspect, anticipated toprovide enhanced therapeutic benefit in humans.

TABLE 1 Maximum Oocyte measured % response 2EVC NR2B potentiation (%)recovered with PAM: EC₅₀ with Ro 25-6981 Ro 25-6981 and (nM) and DCKADCKA

446 Not determined Not determined Compound 1 Comparator Examples from WO2017/100591

1,500 124 91% Example 174

1,190 129 69% Example 181

Solubility Determinations

The solubility of Compound 1 was investigated and compared with Example436 of WO 2017/100591, a constitutional isomer (imidazopyrazinone core)of Compound 1 which demonstrated potency (100% response recovered) inthe Ro 25-6981/DCKA assay. The aqueous solubility of the test compoundwas determined in phosphate buffer saline, pH 7.4, measured byshake-flask method. In this assay, DMSO stock solution of test compoundwas added to buffer followed by equilibration (shaking), filtration, anddetermination of soluble amount by HPLC-UV. As shown in the table,Compound 1 was found to have an approximate 10-fold increase in aqueoussolubility (29 uM vs 3 uM) over the constitutional isomer Example 436.Other imidazopyrazinone comparators from WO 2017/100591 are provided forreference. All comparator analogs are active and potency data isavailable in WO 2017/100591. This improvement is advantageous becauseimproved aqueous solubility, in one aspect, is anticipated to provideenhanced in vivo performance in humans.

TABLE 2 Aqueous solubility (uM)

29 Compound 1 Key Comparator from WO 2017/100591

3 Example 436 Additional Comparators from WO 2017/100591

16 Example 432

26 Example 429

43 Example 430

7 Example 427

20 Example 426

4 Example 432

30 Example 433

5 Example 434

14 Example 435

2 Example 434

7 Example 437

14 Example 445

9 Example 446

69 Example 447

2 Example 438

3 Example 441

91 Example 443

Permeability and Efflux Determinations

Monolayer systems consist of a tight cell layer grown on a poroussupport to separate two fluid compartments. They are widely regarded asthe most sophisticated in vitro tools for medium to high throughputmodeling of important pharmacokinetic barriers, such as intestinalepithelium, blood-brain barrier, etc. (J. Pharm Sci. 2012 April; 101(4):1337-1354). Two systems that are applied widely in monolayer studies arethe human colon carcinoma cell line Caco-2 and MDR1-transfected MDCKIIand LLC-PK1 cells. In monolayer assays, the flux of a compound throughthe monolayer of cells is measured. The unidirectional flux of thecompound of interest is determined by applying it to either the apicalor to the basolateral side of the cell layer and monitoring the timeresolved redistribution of it between the two compartments. Thevectorial transport ratio (often referred to as Efflux Ratio) isdetermined by applying bidirectional measurements [apical-to-basolateral(A-B) and basolateral-to-apical (B-A)]. In general, a ratio higher than2 or lower than 0.5 indicates the contribution of an active transportprocess to the net flux of a compound. In the absence of such transportprocesses this ratio is approximately 1.

TABLE 3 A-B MDCK B-A MDCK Permeability Permeability Efflux (10⁻⁶ cm/sec)(10⁻⁶ cm/sec) Ratio 32.3 32.1 0.99

As shown in Table 3, Compound 1 penetrates membranes well and is notsubject to efflux in the MDCK efflux assay.

Human and Rat Microsome Stability Determinations

The liver is the most important site of drug metabolism in the body.Approximately 60% of marketed compounds are cleared by hepaticCYP-mediated metabolism (McGinnity, D. F.; Soars, M. G.; Urbanowicz, R.A. and Riley, R. J.; Drug Metab. Disp. 32, 1247, (2004)). Livermicrosomes are subcellular fractions which contain membrane bound drugmetabolizing enzymes. Microsomes can be used to determine the in vitrointrinsic clearance of a compound. The use of species-specificmicrosomes can be used to enable an understanding of interspeciesdifferences.

HLM % RLM % Metabolized HLM t_(1/2) HLM mCL_(int) Metabolized RLMt_(1/2) RLM mCL_(int) after 30 min (Min) (μL/min/mg) after 30 min (Min)(μL/min/mg) 34.3 50.8 27.3 1.83 >120 <5

As shown, Compound 1 demonstrates good stability in Human LiverMicrosomes (HLM) and excellent stability in Rat Liver Microsomes (RLM).

In Vivo Clearance

Compound 1 also exhibited improved in vitro microsomal stability and invivo clearance in the rat IV pharmacokinetic study. As shown below, thein vivo clearance of Compound 1 is significantly lower than that ofstructurally-related NMDA PAMs exemplified in WO 2017/100591 for whichIV rat clearance data is available. Of particular note is Example 285, apyrrolopyridinone analog which demonstrated excellent potency in the Ro25-6981/DCKA assay and is exceptionally structurally similar toCompound 1. Rat microsomal stability data is provided as well. Theseimprovements in in vitro and in vivo clearance are advantageous becausereduced clearance, in one aspect, is anticipated to provide enhanced invivo performance in humans.

TABLE 4 Rat liver Oocyte microsome 2EVC NR2B (RLM) In vivo PAM: EC₅₀stability t_(1/2) clearance (nM) (min) (mL/min/kg)

446 >120 7.4 Compound 1 Key Comparator from WO 2017/100591

200 37.8 22.2 Example 285 Additional Comparators from WO 2017/100591

5630 200 19.4 Example 12

589 75.0 23.4 Example 396

463 36.0 26.9 Example 42

726 42 27.8 Example 39

441 60.8 29.4 Example 43

3530 37.8 35.5 Example 9

7010 47.2 43.6 Example 3

1210 37.3 46.2 Example 397

Pharmacology

A. Novel Object Recognition

The cognitive enhancing effect of oral Compound 1 (0.3, 1.0, and 3.0mg/kg) was tested against phencyclidine (PCP)-induced cognitiveimpairment in the rat Novel Object Recognition (NOR) test. Thisexperimental protocol tests for reversal of object recognition memorydeficits caused by chronic administration of the NMDA receptorantagonist, PCP.

Rats were treated twice daily for 7 days with either saline vehicle(control group) or PCP (5 mg/kg, intraperitoneal). Following a 14-daywashout period of no treatment, rats were tested in the NOR paradigm.Briefly, testing involves two sessions (T1 and T2), each lasting 3minutes, 1 hour apart. Vehicle or Compound 1 were administered prior tothe T1 trial. In T1, rats are placed in a test arena with two identicalobjects and allowed to freely explore while their time spent exploringeach object is recorded. In T2, rats are returned to the test arenawhere one of the objects remains the same and the other has beenreplaced with a novel object. The time spent exploring the novel objectversus the time spent exploring the familiar object are recorded andcompared to T1 object exploration times.

Compared to saline control, PCP-treated animals showed a significantreduction in time spent exploring the novel object, confirming aPCP-induced deficit in NOR. As shown by the data in FIG. 1 ,administration of Compound 1 (0.3 and 1.0 mg/kg, oral) significantlyreversed this deficit, as exhibited by an increased novel objectexploration time compared to PCP-treated rats. The 1 mg/kg dose ofCompound 1 fully-reversed the PCP-induced deficit, as the explorationtime at these doses was not significantly different from that in salinecontrol animals.

B. Mismatch Negativity Efficacy Model

Pathophysiological biomarkers of NMDA receptor hypofunction inschizophrenia include EEG measurements of early auditory processingevents, such as “Mismatch Negativity”, an EEG Event-Related Potential(ERP) that measures pre-attentional auditory novelty detection. MMN is atranslatable measure of auditory novelty detection in rats and humans,and is correlated with cognitive and global function in patients withschizophrenia. NMDA receptor antagonists PCP, MK-801, and ketamineelicit acute deficits in MMN in rats (all three NMDAr antagonists) andhuman subjects (ketamine).

Rats implanted with frontal EEG electrodes were presented with differentaudio stimuli comprising an auditory oddball “flip-flop” protocol.Briefly, 1,000 standard tones of 6.0 kHz were delivered at 90%probability, and 100 deviant tones of 8.0 kHz were delivered at 10%probability in pseudo-random order (the flip sequence), and then thissequence was then repeated with the 8 kHz tone as the standard, and the6 kHz tone as the deviant (the flop sequence). MMN is calculated as adifference potential obtained by subtracting the averaged 8 kHz standardtone response (flop) from the averaged 8 kHz deviance tone response(flip) at each 1 ms time point over the EEG recording epochs from 50 msprior to 150 ms after the onset of these 50 ms auditory tones.

In a 4-way crossover design, rats were dosed with vehicle or Compound 1(60 mg/kg) twice, 4 hours apart. Rats were then dosed with salinecontrol or MK-801 (0.2 mg/kg, IP) immediately prior to test sessions.Test sessions were comprised of three 20-minute flip-flop blocks. Asshown by FIG. 2 , administration of vehicle plus MK-801 significantlyimpaired MMN compared to administration of vehicle plus saline. Inaddition, administration of Compound 1 (60 mg/kg BID) preventedimpairment of MMN by MK-801, such that MMN recorded after Compound 1plus MK-801 was no longer different from MMN recorded after vehicle andsaline, and was significantly larger when compared to MMN recorded afteradministration of vehicle and MK-801.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

The invention claimed is:
 1. A method for treating Alzheimer's Diseasein a subject comprising administering to said subject a therapeuticallyeffective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. A method for treatingAlzheimer's Disease in a subject comprising administering to saidsubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound of the formula:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.